Cystic fibrosis (CF) is the most common lethal genetic disease in the Caucasian population. It is caused by mutations in the CF gene, encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel. The most prevalent CF mutation, deletion of phenylalanine 508 (?F508) impairs the posttranslational folding, gating characteristics, and the biosynthetic and endocytic processing of CFTR. The functional expression defect of CFTR at the plasma membrane leads to impaired chloride, bicarbonate and fluid transport in secretory epithelia, manifesting in recurrent lung infection, the primary cause of mortality in CF. A major focus of CF research is the identification of small-molecule correctors of defective CFTR processing and potentiators of defective gating that target that the ?F508 and other CFTR mutations. The efficacy of the best correctors available (VX-809) is low, such that treated cells show only ?15% of chloride conductance compared to non-CF cells. We and others have demonstrated that chronic exposure to gating potentiators (e.g. VX-770) destabilizes ?F508-CFTR and reduces correction efficacy. Phase III clinical data, indeed, showed only modest but significant clinical efficacy of combination VX-809 and VX-770 therapy. This competitive renewal builds on our recent discoveries of: a) corrector and potentiator molecules by high throughput screening (HTS) assays; b) the correction requirement of both primary folding defects of ?F508-CFTR, including destabilization of the NBD1 conformation and the NBD1-MSD2 interface in the context of cooperative domain folding; c) preliminary identification of novel potentiator molecules that do not destabilize ?F508-CFTR; and d) correction of the functional expression defect of the W128X-CFTR nonsense mutation synergistically by corrector and potentiator combination. To identify corrector-potentiator combinations that restore ?F508-CFTR folding and chloride channel function to >50% of its wild-type counterpart, we propose to identify and validate efficacious potentiators that lack destabilizing effect of the ?F508-CFTR in Aim 1. In Aim 2 we will utilize novel localized structure defect-targeted (LSDT) screening approaches to identify distinct, structure-specific correctors as pharmacological chaperones that stabilize NBD1 and act synergistically with interface stabilizing drugs (e.g. VX-809). Engineered primary airways epithelial cells will increase the success rate of these screens. The mechanism of action of novel correctors will be established by biophysical, biochemical and cell biological assays. Based on the results of Aim 1-2, in Aim 3 novel mechanistic studies on the W1282X-CFTR functional expression defect, mutation-specific biochemical and functional HTS assays will be implemented to identify small-molecule correctors and potentiator and establish their mechanism of action.